Title: The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 4 Monooxygenation
1The Organic Chemistry of Enzyme-Catalyzed
Reactions Chapter 4 Monooxygenation
2Monooxygenation
Table 4.1. Typical reactions catalyzed by
monooxygenases
3Reaction catalyzed by lactate oxidase from
Mycobacteria
Internal Monooxygenase
Flavin-dependent Hydroxylases
Scheme 4.1
No external reducing agent required
4The lactate oxidase reaction under anaerobic
conditions
One Turnover Experiment (enzyme concentration in
excess over substrate)
Acting like an oxidase
Scheme 4.2
5Reaction of Reduced Lactate Oxidase with Pyruvate
and Oxygen
Scheme 4.3
If O2 is added first, then 14Cpyruvate,
pyruvate is unchanged and H2O2 is formed.
Therefore, pyruvate is an intermediate.
Model study
6Possible Mechanisms for Lactate Oxidase
like DAAO
flavin hydroperoxide acts as a nucleophile
electrophilic substrate
Scheme 4.4
7NAD(P)H reduction of flavin
External Monooxygenases
O2 activation
Scheme 4.5
Activated O2 is probably in the form of flavin
hydroperoxide
8Mechanism proposed for flavin-dependent
hydroxylases
Nucleophilic Substrates
stopped-flow spectroscopic evidence for boxed
intermediates
flavin hydroperoxide acts as electrophile
electrophilic aromatic substitution
Scheme 4.6
9Hammett Study
p-hydroxybenzoate hydroxylase
log Vmax for hydroxylation vs pKa linear free
energy relationship ? -0.5
(Electron deficient mechanism)
Consistent with electrophilic aromatic
substitution
10Reaction catalyzed by bacterial luciferase
Electrophilic Substrates
long-chain aldehydes (electrophilic substrates)
Scheme 4.7
11Nucleophilic Mechanism for Bacterial Luciferase
on warming
isolated by cryoenzymology (-30 ?C in mixed
aqueous-organic media)
electrophilic substrates
Scheme 4.8
detected spectro-photometrically
However, with 8-substituted FMN analogues rate
increases with decreasing one electron oxidation
potentials of analogues
12Chemically Initiated Electron Exchange
Luminescence (CIEEL) Mechanism for Bacterial
Luciferase
SET
Scheme 4.9
13Dioxirane mechanism for bacterial luciferase
Alternative One-electron Mechanism via a Dioxirane
SET
kx/kh vs. ?p for 8-substituted flavins
? -4
(facilitated by e- donation)
Scheme 4.10
Inconsistent with Baeyer-Villiger mechanism (?
values 0.2 to 0.6)
14Baeyer-Villiger Oxidation of Ketones
Scheme 4.11
Migratory aptitude - more e- donating group
migrates (in the case above, R)
15Reaction catalyzed by cyclohexane oxygenase
Ketone Monooxygenases - an Example of a
Baeyer-Villiger Oxidation
Scheme 4.12
C4a-FAD hydroperoxide intermediate detected
16Other Reactions Catalyzed by Cyclohexanone
Oxygenase
same migratory aptitudes as nonenzymatic reaction
Scheme 4.13
17Cyclohexane Oxygenase Proceeds with Retention of
Configuration (like nonenzymatic)
Scheme 4.14
18Migratory Aptitude of Cyclohexanone
Oxygenase-catalyzed Reaction
Same migratory aptitude as nonenzymatic (3 gt 2
gt 1 gt Me)
Scheme 4.15
19no loss of D (like nonenzymatic reaction)
20Baeyer-Villiger-type Mechanism Proposed for
Cyclohexanone Oxygenase
electrophilic substrate
Scheme 4.16
21Reaction of Cyclohexanone Oxygenase with Boranes
same as nonenzymatic reaction
Scheme 4.17
22Reactions Catalyzed by Ketone Monooxygenase
when R1 R2 Me 1 20 R1 H
R2 Me 1 1
(same as nonenzymatic reaction)
Scheme 4.18
23Reactions Catalyzed by the Ketone Monooxygenase
from A. calcoaceticus
1S
5R
1R
5S
gt95 ee
gt95 ee
racemate
Scheme 4.19
24Reactions Catalyzed by the Ketone Monooxygenase
from P. putida
1R
5S
1S
5R
50 ee
gt95 ee
gt95 ee
racemate
Scheme 4.20
25Pterin-dependent Monooxygenasesaromatic
hydroxylation
pteridine ring
26Tetrahydrobiopterin
- Fe2 also required for activity
- Only a few enzymes require tetrahydrobiopterin
- Important in biosynthesis of dopa,
norepinephrine, epinephrine, and serotonin - Reactions similar to flavoenzymes
27Comparison of the Dihydrobiopterin and
Tetrahydrobiopterin with Oxidized Flavin and
Reduced Flavin
Scheme 4.21
28Reaction Catalyzed by Phenylalanine Hydroxylase
NIH shift 1,2 migration
Scheme 4.22
Similar to flavin hydroxylases except 2H washed
out with flavoenzymes
29Possible Intermediate
30Mechanism of the Reaction Catalyzed by
Tetrahydrobiopterin-dependent Monooxygenases
nucleophilic substrate
discussed with heme-dependent enzymes
Scheme 4.23
31Reaction of dihydrophenylalanine with
phenylalanine hydroxylase
Evidence for Arene Oxide Intermediate
Scheme 4.24
32Arene Oxide Mechanism Proposed for
Tetrahydrobiopterin-dependent Monooxygenases
Tyr
Scheme 4.25
m-Tyr
Incubation with 4-2HPhe should favor formation
of
m-Tyr (isotope effect),
and 3,5-2H2Phe should favor
Tyr, but they do not.
Therefore, not an arene oxide intermediate
33Cationic Mechanism Proposed for
Tetrahydrobiopterin-dependent Monooxygenases
Fe
as X is larger
Scheme 4.26
m-Tyr
The larger the size of X, the more m-Tyr product
34Alternative Species
These species could account for alkyl
hydroxylation products (heme chemistry), e.g. with
hydroxylation here
35Heme-Dependent Monooxygenases
Heme
Cytochrome P450s (gt500 different isozymes)
require NAD(P)H and O2
Protection from xenobiotics
36Reactions Catalyzed by Heme-dependent
Monooxygenases
-
-
37Molecular Oxygen Activation by Heme-dependent
Monooxygenases
(requires NADPH)
In P450cam Thr-252
low-spin state
high-spin state
FeIII more readily accepts e-
cytochrome P450 reductase
calculations favor this structure
Scheme 4.27
means isolated and characterized
38Alkane Hydroxylation
Two-step radical mechanism with oxygen rebound
for alkane oxygenation by heme-dependent
monooxygenases
3 gt 2 gt 1
retention of configuration
Scheme 4.28
(suggests C-H cleavage is not the
rate-determining step)
Intermolecular isotope effect lt 2
C-H cleavage during catalysis
Intramolecular isotope effect gt 11
39Products from the Reaction of all
Exo-2,3,5,6-tetradeuterionorbornane with the
CYP2B4 Isozyme of Cytochrome P450
Scheme 4.29
Scrambling of stereochemistry supports 2-step
radical mechanism
40Radical clock approach for determination of
reaction rates in radical rearrangement reactions
Radical Clocks - detection of radical
intermediates
known
Scheme 4.30
The rate of hydroxylation can be calculated
(lifetime of radical intermediate)
41Cytochrome P450-catalyzed monooxygenation of a
cyclopropane analogue
Example of Radical Clock
a
Scheme 4.31
b
From kr 2 ? 109 s-1 and the ratio of a/b, can
calculate kOH 2.4 ? 1011 s-1
42Cytochrome P450-catalyzed Oxidation of
Trans-1-methyl-2-phenylcyclopropane
Scheme 4.32
Perdeuteration (CD3) gives
increased pathway b called metabolic switching
43Another ultrafast radical clock reaction
catalyzed by cytochrome P450
Evidence against a True Radical Intermediate
3 ? 1011 s-1
Scheme 4.33
very little
kOH has to be faster than the decomposition of a
TS (6 ? 1012 s-1) therefore propose carbocation
after oxidation step
44A Hypersensitive Radical Probe Substrate to
Differentiate a Radical from a Cation
Intermediate Generated by Cytochrome P450
Scheme 4.34
based on nonenzymatic reactions
With CYP2B1 - mostly unrearranged, but small
amount of both 4.48 and 4.51 therefore radical
lifetime is 70 fs
45A Concerted, but Nonsynchronous, Mechanism
Proposed for Cytochrome P450
Scheme 4.35
General conclusion More than one oxidizing
species involving more than one pathway with
multiple high-energy heme complexes (radical and
cation)
46Two-step radical mechanism with oxygen rebound
for alkene oxygenation by heme-dependent
monooxygenases
Alkene Epoxidation
lifetime?
Scheme 4.36
47Cytochrome P450-catalyzed epoxidation of
trans-1-phenyl-2-vinylcyclopropane
Evidence for Short-lived Radical
only
cyclopropyl/carbinyl radical rearrangement not
detected
Scheme 4.37
48Cytochrome P450-catalyzed formation of an arene
oxide
Arene Hydroxylation Isolation of first arene oxide
Scheme 4.38
Is it an intermediate or side product?
49A common intermediate in the oxygenation of
naphthalene
Evidence for a Cyclohexadienone Intermediate
either
same product and 2H incorporation from both
isomers
Should have observed 1- and 2- hydroxynaphthalene
because of an isotope effect
Scheme 4.39
50Concerted (pathway a) and Stepwise (pathway b)
Mechanisms for the Potential Conversion of an
Arene Oxide to a Cyclohexadienone
concerted
stepwise
Scheme 4.40
Evidence against concerted 1) no deuterium
isotope effect 2) Hammett plot shows large
-?
(carbocation intermediate)
51Mechanism proposed for heme-dependent oxygenation
of aromatic compounds
Isotope Effect and Hammett Studies Indicate
either Radical or Cation (or both) Intermediates,
but not Arene Oxide
reasonable
unfavorable
NIH shift
favorable
Electrophilic addition when R is o/p directing,
get mostly p product when R is m-directing, get m
and p products
Scheme 4.41
52Electron transfer mechanism proposed for
heme-dependent oxygenation of sulfides
Sulfur Oxygenation
Scheme 4.42
Linear free energy relationship log kcat vs.
one-electron oxidation potential as well as ?
53N-Dealkylation
Electron transfer mechanism proposed for
heme-dependent oxygenation of tertiary amines
Scheme 4.43
With primary and secondary amines hydrogen atom
abstraction mechanism favored (see next slide)
54O-Dealkylation
Hydrogen atom abstraction mechanism proposed for
heme-dependent oxygenation of ethers
Scheme 4.44
Not electron transfer mechanism--
oxidation potential for oxygen is too high
55Reaction catalyzed by aromatase
C-C Bond Cleavage
androstenedione
estrone
Scheme 4.45
56Fate of the Atoms during Aromatase-catalyzed
Conversion of Androstenedione to Estrone
also a substrate
also a substrate
Scheme 4.46
First two oxygenation steps proceed by normal
heme hydroxylation mechanism
57Three Possible Mechanisms for the Last Step in
the Aromatase-catalyzed Oxygenation of
Androstenedione
heme peroxide
like Fl-OO- addition to aldehydes
Scheme 4.47
58Oxidation of pregnenolone, catalyzed by an
isozyme of cytochrome P450 (P45017?)
Evidence for Heme Peroxide Mechanism
retained
Scheme 4.48
FeIV-O would have abstracted a C21 CH3 hydrogen
or a C16 or C17 H
59Hydrogen Atom Abstraction Mechanism, Using a Heme
Iron Oxo Species, for the P45017?-catalyzed
Oxygenation of Pregnenolone
Scheme 4.49
retained
In 2H2O, ketene would give H2C2HCOO2H no 2H
found in CH3 group of acetate, therefore not
FeIV-O
60Evidence for a Nucleophilic Mechanism, Using Heme
Peroxy Anion Followed by a Radical Decomposition
of the Heme Peroxide, for the P45017?-catalyzed
Oxygenation of Pregnenolone
Scheme 4.50
Mutation of Thr-302 (T302A) in P450 2B4 (needed
for formation of iron oxo species) decreased
hydroxylation activity, but increased deacylation
(nucleophilic) activity
61Nucleophilic mechanism, using heme peroxy anion
followed by a Baeyer-Villiger rearrangement, for
the lanosterol 14?-methyl demethylase-catalyzed
oxygenation of lanosterol
Further Evidence for Heme Peroxide
Scheme 4.51
isolated, also a substrate
62Nucleophilic Mechanism, Using Heme Peroxy Anion,
Followed by a Radical Decomposition of the Heme
Peroxide, for the Lanosterol 14?-Methyl
Demethylase-catalyzed Oxygenation of Lanosterol
Would Not Give the Baeyer-Villiger Product
No formate ester formed
Scheme 4.52
63Synthesized to test Baeyer-Villiger mechanism
with aromatase - no estrone Maybe aromatase and
P45017? have different mechanisms from that of
lanosterol 14?-methyl demethylase
64Model Studies on the Mechanism of Aromatase
gives aromatase product
Scheme 4.53
65Mechanism proposed for aromatase initiated by
dienol formation
Revised Aromatase Mechanism
Scheme 4.54
66Nonheme Iron Oxygenation Methane monooxygenases
binuclear iron cluster
CH4 ? CH3OH
67Binuclear Ferric Cluster of Methane Monooxygenase
Scheme 4.55
soluble methane monooxygenase
XAS and Mössbauer spectroscopy support 4.83a, not
4.83b Studies with the hypersensitive
cyclopropane probe (4.46, Scheme 4.34) and
methylcubane indicate a cation, not radical,
intermediate Therefore mechanism like P450
68Reaction catalyzed by dopamine ?-monooxygenase
Copper-dependent Oxygenation
from ascorbic acid
Scheme 4.56
Optimal activity with 2 CuII per subunit one
CuII catalyzes e- transfer from ascorbate one
CuII catalyzes oxygen insertion into substrate
69Mechanism Proposed for Dopamine ?-Monooxygenase
H
Scheme 4.57
Hammett plot ? -1.5
fits better to ?? than ??, suggesting a radical
with a polar TS